Fluorescent compounds

ABSTRACT

Fluorescent dye compounds of formula (I) are disclosed. These compounds are useful as they interact with organic compounds in a manner such that excitation with certain wavelengths of light results in fluorescent emmission. Detection and/or monitoring of the fluorescence provides a means for the detection or quantification of organic compounds when bound to these fluorescent dye compounds. Formula (I), wherein: each of R, R′ and R″ is a hydrogen atom, halogen atom or a straight or branched chain (C 1-20 alkyl, alkenyl or alkynyl group optionally substituted with one or more halogen, hydroxy and/or oxy group: rings A, B and C optionally include one or more double bonds; rings B and C are optionally substituted with one or more halogen atoms.

TECHNICAL FIELD

[0001] This invention relates generally to novel fluorescent compoundsand to compounds which can function as fluorescent dyes, methods ofisolating such compounds from microorganisms and uses of such compoundsin scientific applications.

BACKGROUND ART

[0002] Compounds that fluoresce have many uses and are known to beparticularly suitable for biological applications where fluorescence isrequired for the detection of whole cells, cellular components, andcellular functions. For example, many diagnostic and analyticaltechniques require the samples to be fluorescently tagged so that theycan be detected. This is achieved by using fluorescent dyes or probeswhich interact with a wide variety of materials such as cells, tissues,proteins, antibodies, enzymes, drugs, hormones, lipids, nucleotides,nucleic acids, carbohydrates, or natural or synthetic polymers to makefluorescent conjugates.

[0003] With synthetic fluorescent probes, ligands are frequently used toconfer a specificity for a biochemical reaction that is to be observedand the fluorescent dye provides the means of detection orquantification of the interaction. These applications include, amongothers, the detection of proteins (for example in gels or aqueoussolution), cell tracking, the detection of proteins via fluorescentlylabelled antibodies, the assessment of enzymatic activity, the stainingof nucleic acids.

[0004] Long wavelength absorbance usually increases the utility of afluorescent probe since it reduces the interference from cellularauto-fluorescence and reduces the cytotoxic effect of the fluorophore incombination with light. Although lasers are particularly useful as aconcentrated light source for the excitation of fluorescence, at presentthe output of lasers is restricted to particular wavelengths of light.Compounds whose excitation spectra coincide with laser output aretherefore of high utility. The argon laser is the most common lightsource for excitation of fluorescence, and has principal outputs, lightat 488 nm and 514 nm. Fluorescent compounds that are excited by eitherof these wavelengths are therefore of particular utility. Alternatively,excitation of fluorescence can be achieved using solid state lightsources such as Light emitting diodes. Fluorescent compounds excited bythe light emitted from these alternative sources are also of particularutility.

[0005] Red fluorescent compounds are used extensively in many fields ofbiological study. Many of these, including Texas red, Tetramethylrhodamine-isothiocyanate or red emitting BODIPY dyes require excitationat green wavelengths such as 542 nm. This limits their use in manyapplications, especially those where the argon-ion laser is used forexcitation. Compounds such as ethidium bromide, can be excited withlight from the argon-ion laser (520 nm band), but are not generallysuitable for tagging of organic molecules other than nucleic acids.Other compounds such as phycoerythrin, can be excited using theargon-ion laser (488 nm) and does emit in the orange/red wavelengths.Phycoerythrin, however, has poor stability and a high molecular weightmaking it unsuitable for many applications such as cell tracking,labelling of nucleic acids or staining proteins.

[0006] For staining of proteins, there are a number of methodsavailable. These methods can utilise non-fluorescent compounds, orfluorescent compounds. The most commonly used method utilises Coomassieblue which is non-fluorescent, can require the use of large amounts oforganic solvents and is time consuming. Other fluorescence-basedprotein-detection methods are available which are potentially moresensitive than non-fluorescent methods. However, these methods are ingeneral much more expensive than non-fluorescent methods which limitstheir widespread use. Therefore, compounds that combine useful spectralcharacteristics, and relatively high sensitivity will be of particularutility.

[0007] There are several methods for the quantification of protein insolution. These methods are based on a range of techniques, and includemethods where dyes bind to soluble proteins. These dyes can be eithernon-fluorescent or fluorescent compounds. Fluorescent dye-based methodsare often more sensitive than the non-fluorescent dyes, and allow forthe determination of protein concentration over a wide range ofconcentrations. Compounds that combine useful spectral characteristicswith an ability to bind proteins will be of particular utility.

[0008] In enzymatic studies, there is widespread use of fluorescentcompounds for the detection of particular enzymatic activities. Forexample, fluorescein di-β-D-galactopyranoside (FDG) is a non-fluorescentcompound that is sequentially hydrolysed by the enzyme β-galactosidasefirst to generate fluorescein monogalactoside and then to fluoresceinwhich is highly fluorescent. The cleavage of the FDG compound can bemonitored by the increase in fluorescence in the solution, and thusallows sensitive quantification of enzymatic activity. At present, onlya limited number of fluorophores are suitable for this procedure.Therefore, novel fluorescent compounds that can be conjugated to avariety of substrates will be of utility.

[0009] For dual colour staining, there is a very limited choice of lowmolecular weight fluorophores. The predominant green fluorophore isfluorescein, which strongly absorbs light from the 488 nm band of theargon ion laser, and re-emits at 518 nm. At present there are fewcompatible red or orange fluorophores that are of low molecular weightand are excited by the 488 nm or 514 nm bands of the argon ion laser.Therefore, low molecular weight compounds that are excited by argon ionlasers and emit at wavelengths greater than 600 nm will be of utility,particularly if there is minimal spectral overlap with fluorescein.

[0010] The present inventors have isolated new compounds derived from afungus that is capable of combining readily with a range of organicmolecules to produce fluorescent complexes.

DISCLOSURE OF INVENTION

[0011] In a first aspect of the invention, there is provided a compoundaccording to formula (I):

[0012] wherein:

[0013] each of R, R′ and R″ is a hydrogen atom, halogen atom or astraight or branched chain C₁₋₂₀ alkyl, alkenyl or alkynyl groupoptionally substituted with one or more halogen, hydroxy and/or oxygroup;

[0014] rings A, B and C optionally include one or more double bonds;

[0015] rings B and C are optionally substituted with one or more halogenatoms; and

[0016] the compound is capable of interacting with an organic compoundand when interacting with the organic compound, the compound emitsfluorescence after excitation at a broad range of wavelengths.Preferably, the compound is excited at wavelengths in the range of300-560 nm, more preferably, 380-530 nm and even more preferably, UVwavelengths and/or blue wavelengths. Preferably the compound emits inthe wavelengths of 460 to 700 nm. More preferably its emission peak iscentered around 530 nm when interacting with an organic compound such assodium dodecyl sulphate and centered around 605 nm when interacting witha biomolecule such as a protein or cell.

[0017] Preferably, the first aspect of the invention provides a compoundaccording to formula (Ia):

[0018] Preferably, the compound according to formula Ia has thestereochemistry as depicted in formula (Ib):

[0019] Preferably, R is a straight or branched chain C₁₋₂₀ conjugatedalkenyl group optionally substituted with hydroxy and oxy groups; R′ isa straight or branched chain C₁₋₂₀ alkyl group optionally substitutedwith a hydroxy group and R″ is a straight or branched chain C₁₋₂₀ alkylgroup.

[0020] More preferably, R=—C(OH)CHC(O)(CH)₆CH₃, R′=—CH₂OH and R″=Me suchthat the present invention according to the first and second aspectsconsists in the compound 5,6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dioneaccording to formula (Ic):

[0021] In a second aspect, the present invention consists in a compoundaccording to formula (I), wherein each of R, R′ and R″ is a hydrogenatom, halogen atom or a straight or branched chain C₁₋₂₀ alkyl, alkenylor alkynyl group optionally substituted with one or more halogen,hydroxy and/or oxy groups;

[0022] ring A, B and C are optionally substituted with one or morehalogen atoms; and

[0023] rings A, B and C optionally include one or more double bonds;with the proviso that:

[0024] (i) R≠—C(OH)CHC(O)(CH)₆C(Et)(Me) when R′=—CH₂OH, R″=Me, ring Aincludes a double bond between C3 and C3a; ring B includes a double bondbetween C4 and C4a; and ring C includes a double bond between C8 andC8a;

[0025] (ii) R≠—C(OH)CHC(O)(CH)₆C(Et)(Me) when R′=Me, R″=Me, ring Aincludes a double bond between C3 and C3a; ring B includes a double bondbetween C4 and C4a; and ring C includes a double bond between C8 andC8a;

[0026] (iii) R≠—C(OH)CHC(O)(CH)₆C(Et)(Me) when R′=Me, R″=Me, ring Aincludes a double bond between C3 and C3a; ring B includes a double bondbetween C4 and C4a; and ring C includes a double bond between C8 and C8aand C5 and C6;

[0027] (iv) R≠—C(O)(CH)₄Me when R′=-n-propyl, R″=Me, ring A includes adouble bond between C3 and C3a; ring B includes a double bond between C4and C4a; and ring C includes a double bond between C8 and C8a and C5 andC6;

[0028] (v) R≠—C(O)(CH)₆Me when R′=-n-propyl, R″=Me, ring A includes adouble bond between C3 and C3a; ring B includes a double bond between C4and C4a; and ring C includes a double bond between C8 and C8a and C5 andC6;

[0029] (vi) R≠—C(O)(CH)₅Me when R′=—(CH)₂Me, R″=Me, ring A includes adouble bond between C3 and C3a; ring B includes a double bond between C4and C4a; and ring C includes a double bond between C8 and C8a and C5 andC6;

[0030] (vii) R≠—C(O)(CH)₆Me when R′=—(CH)₂Me, R″=Me, ring A includes adouble bond between C3 and C3a; ring B includes a double bond between C4and C4a; and ring C includes a double bond between C8 and C8a and C5 andC6;

[0031] (viii) R≠—(CH)₂C(Me)CHC(Me)(Et) when R′=—Ac, R″=Me, C4=Cl, ring Aincludes a double bond between C3 and C3a; ring B includes a double bondbetween C4 and C4a; and ring C includes a double bond between C8 and C8aand C5 and C6;

[0032] (ix) R≠—(CH)₂C(Me)CHC(Me)(Et) when R′=—Ac, R″=Me, ring A includesa double bond between C3 and C3a; ring B includes a double bond betweenC4 and C4a; and ring C includes a double bond between C8 and C8a and C5and C6;

[0033] (x) R≠—(CH)₂C(Me)CH-i-Pr when R′=—Ac, R″=Me, ring A includes adouble bond between C3 and C3a; ring B includes a double bond between C4and C4a; and ring C includes a double bond between C8 and C8a and C5 andC6;

[0034] (xi) R≠—C(O)(CH)₄Me when R′=—(CH)₂Me, R″=Me, ring A includes adouble bond between C3 and C3a; ring B includes a double bond between C4and C4a; and ring C includes a double bond between C8 and C8a and C5 andC6;

[0035] (xii) R≠—C(O)(CH)₄Me when R′=-n-Pr, R″=Me, ring A does notinclude a double bond; and rings B and C include double bonds betweenC8a and C4a and C5 and C6;

[0036] (xiii) R≠—C(O)(CH)₄Me when R′=-n-Pr, R″=Me, ring A, B and C donot include double bonds;

[0037] (xiv) R≠—C(O)(CH)₆,Me when R′=—(CH)₂Me, R″=Me, ring A does notinclude a double bond; and rings B and C include double bonds betweenC8a and C4a and C5 and C6;

[0038] (xv) R≠—C(O)(CH)₄Me when R′=—C(CH₂)(Me), R″=Me, ring A does notinclude a double bond; and rings B and C include double bonds betweenC8a and C4a and C5 and C6;

[0039] (xvi) R≠—C(O)(CH)₄Me when R′=—(CH)₂Me, R″=Me, ring A does notinclude a double bond; and rings B and C include double bonds betweenC8a and C4a and C5 and C6.

[0040] Preferably, the compound according to second aspect of theinvention is in accordance with formula (Ia) and more preferably, hasthe stereochemistry as depicted in formula (Ib).

[0041] Even more preferably, R=—C(OH)CHC(O)(CH)₆CH₃, R′=—CH₂OH and R″=Mesuch that the present invention according to the second aspect consistsin the compound 5,6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dioneaccording to formula (Ic).

[0042] It will be appreciated that the compound according to formula(I)-(Ic) includes all corresponding tautomeric structures.

[0043] Chemically, the compounds of formula (I)-(Ic), such as5,6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione,are members of the azaphilone compounds. The azaphilone compounds areproduced by a variety of fungi, and have been investigated for theirantibiotic functions, their ability to inhibit enzymatic function, andtheir role as colouring agents in the food additives produced byMonascus sp.

[0044] The azaphilone nucleus has also been found in the pigmentsproduced by Monascus sp. The pigments containing the common azaphilonecore include dihydro-monascin and monascorubin. There is no record oftheir utility as fluorescent dyes for the staining of biomolecules ororganic compounds.

[0045] None of the prior art known to the applicant teaches or suggeststhat a compound of the structure described in formula (I)-(Ic) would befluorescent, nor suggests that it would be suitable for use in thefluorescent staining of bio-molecules or organic compounds. There areaccordingly unexpected and advantageous properties associated with thiscompound including sensitive detection of proteins in gels, celltracking and dual colour staining.

[0046] The present inventors have unexpectedly found that compoundsaccording to formula (I)-(Ic) are not fluorescent in aqueous solution,but are capable of interacting with organic compounds to produce anintensely fluorescent stain. In a preferred embodiment, the compoundsaccording to formula (I)-(Ic) of the invention are used in the detectionand tagging of organic molecules.

[0047] Preferably, the compound according to formula (I)-(Ic), whenbound to an organic molecule emits fluorescence after excitation at bluewavelengths.. More preferably, the compounds according to formula(I)-(Ic) are excited by light in the absorbance range 300-560 nm.

[0048] In a preferred embodiment, a compound according to formula(I)-(Ic) interacts with proteins to produce a fluorescent complex thatcan be excited by light generated by standard UV transilluminators (307nm). Upon excitation, the fluorescent complex emits light over a widerange of wavelengths allowing the protein complexes to be detected. Theexcitation wavelength of the protein/dye complex includes that ofethidium bromide complexed with DNA (Absorption maximum 518 nm, Emissionmaxima 605 nm). This is of particular utility because it allows the sameequipment to be used to detect both DNA and protein in gels. The broadrange of excitation wavelengths allows the compound to be excitedstrongly by 488 nm and 514 nm bands of the argon ion laser, as well asabsorb light emitted from diode light sources such as those the emit ataround 400 nm

[0049] In another preferred embodiment, a fluorescent form of thecompound according to formula (I)-(Ic) is capable of strongly absorbinglight from the 488 nm output of the argon-ion laser, and to re-emit thelight at wavelengths longer than 600 nm.

[0050] In another preferred embodiment, the compound according toformula I is included in a composition, along with an analyticallyacceptable carrier, and may be used in combination with a fluorochromesuch as fluorescein in dual staining applications. Such a fluorochromemay be included in the composition or separately used.

[0051] The compound according to formula (I)-(Ic) may be produced by afungal species. Preferably, the fungal species is the strain depositedat the Australian Government Analytical Laboratories (AGAL) on Jan. 15,1998 identified by Accession Number NM98/00507.

[0052] The methods for purification, isolation, as well of the structureof the novel fluorophore 5,6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dioneand related compounds have not previously been described.

[0053] The binding of the compound according to formula (I)-(Ic) toorganic molecules may involve direct chemical or physical binding or maybe achieved by the use of “linking molecules” known to the art. Organicmolecules which may be bound by the compound of the invention when usedas a fluorescent dye include, for example, proteins, peptides, sugars,nucleic acids, antibodies, cell surface biomolecules, detergents andcells. Due to the compound's fluorescent and organic compound-bindingcharacteristics, it will be appreciated that the compound will have usein any application where detection of a fluorescent dye attached to anorganic compound is required.

[0054] In a third aspect, the present invention consists in a process ofproducing a compound according to the first and second aspect of theinvention, the process comprising culturing a fungal species underconditions such that the fungal species produces the compound; andseparating the compound from the culture.

[0055] The compound according to formula (I)-(Ic) can be used as afluorescent dye when in a crude culture extract or when purified byextraction and separation techniques.

[0056] It has been found by the present inventors that after inoculationand incubation of the strain identified by AGAL Acession NumberNM98/00507 on the growth medium, a compound which is suitable as afluorescent dye is produced by the fungus. It will be appreciated thatit should be possible to produce compounds according to formula (I)-(Ic)of the first and second aspect of the present invention by usingtechniques other than from the supernatant of a suitable fungal culture.For example, if the fungus produces a “pre-cursor”, then it will bepossible to modify that pre-cursor to its fluorescent form by chemical,physical or enzymatic means. The knowledge that such compounds can beobtained from microorganisms should allow the discovery and productionof other compounds suitable for use as fluorescent dyes belonging to thesame family or quite distinct compounds with useful characteristics. Thecompound according to formula I may also be produced synthetically bydirect chemical synthesis, or by modification of intermediate(s) in thebiosynthetic pathway used by the fungi.

[0057] The compound according to formula (I)-(Ic) of the presentinvention may also be produced synthetically by chemical means. Theknowledge that a new fluorescent compound is produced by fungi may leadto other means of producing the compound apart from culturing the fungiunder the required conditions.

[0058] The compound according to formula (I)-(Ic) of the presentinvention has the distinct advantage that it binds to cells and otherorganic molecules in its fluorescent form so can be used as a means totrack the cells or the other organic molecules when labelled with thecompound.

[0059] In a fourth aspect, the present invention consists in use of thecompound according to the first and second aspect of the presentinvention as a fluorescent dye or marker, preferably in scientifictechniques for staining, labelling and/or detecting organic molecules.

[0060] Examples of the use of the compound according to the first andsecond aspect of the present invention include but are not restricted tocell tracking dyes for microscopy, membrane fluidity dyes, conjugationwith antibodies, conjugation to nucleic acids, cell surface ligandimaging dyes, conjugation to sugars, cytometric analysis, and confocalmicroscopy. It will be appreciated, however, that the compound would besuitable for any use where fluorescence in the red wavelengths isrequired, including when excited at 488 nm.

[0061] In a fifth aspect, the present invention consists in a method offluorescent-labelling an organic compound, the method comprising causingthe compound according to the first or second aspect of the presentinvention to bind to an organic compound such that the organic compoundis fluorescently labelled with the compound.

[0062] Preferably, the fluorescently labelled organic compound isdetected when exposed to a wide range of wavelengths, preferablywavelengths in the range of 300-560 nm.

[0063] The organic compound may include, but is not limited to,proteins, peptides, sugars, nucleic acids, antibodies, cell surfacebiomolecules, detergents and cells. The compound may bind directly tothe organic compound due to a chemical or physical association or maybind to the organic compound via a linking molecule. If the compound isattached to a ligand specific for the organic compound, for example anantibody or lectin, then the binding of that ligand to the organiccompound will cause the organic compound to be fluorescently labelled.

[0064] In a sixth aspect, the present invention consists in a method ofdetecting a organic compound in a sample including the organic compound,the method comprising labelling the organic compound according to themethod of the fifth aspect of the present invention; and detecting theorganic compound in the sample by monitoring or detecting itsfluorescence. Preferably, the labelled organic compound is detected whenthe sample is exposed to light from a wide range of wavelengths,preferably wavelengths in the range of 300-560 nm, more preferably bluewavelengths.

[0065] The monitoring or detecting of the fluorescence of thelabelled-organic compound may be by any means known to the art. Suchmeans include, but not limited to, transillumination, spectroscopymicroscopy and cytometry.

[0066] Throughout this specification the word “comprise”, or variationssuch as “comprises” or “comprising”, will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

[0067] Any discussion of documents, acts, materials, devices, articlesor the like which has been included in the present specification issolely for the purpose of providing a context for the present invention.It is not to be taken as an admission that any or all of these mattersform part of the prior art base or were common general knowledge in thefield relevant to the present invention as it existed in Australiabefore the priority date of each claim of this application.

[0068] In order that the present invention may be more clearlyunderstood a preferred forms will be described with reference to thefollowing examples and accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0069]FIG. 1 shows an NMR spectrum of 5,6-dihydro-3-[(1Z, 4E, 6E,8E)-1hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dionea fluorescent compound according to the first and second aspects of theinvention.

[0070]FIG. 2 shows an example of an emission spectra of the compoundidentified by FIG. 1 when bound to bovine serum albumin (BSA) andpropidium iodide bound to DNA. The number of moles of the purifiedcompound used to bind to excess BSA, were equal to the number of molesof propidium iodide used to bind to excess DNA. There are two excitationbands resulting in emission at 605 nm. As seen, excitation by light at390-400 nm results in maximum emission of light at 605 nm.

MODES FOR CARRYING OUT THE INVENTION Example 1

[0071] Production of Extract A.

[0072] A biologically pure culture of the micro-organism having all ofthe identifying characteristics of the strain identified by AGALAccession Number NM 98/00507 was obtained.

[0073] A growth medium for the fungus, AGAL Accession Number NM98/00507, was prepared by adding 40 g Sucrose (CSR), 5 g Yeast Extract(Difco), 10 g Peptone (Difco) and 10 g Agar (Difco) to 1 L of water. Themixture was autoclaved for 15 min at 115° C. to both sterilise themedium and to dissolve the agar. The liquid was poured into culturedishes and allowed to set at room temperature. After cooling, a cultureof the fungus was inoculated onto the surface of the medium, andincubated at 25° C. for three days. The culture was transferred to arefrigerator and incubated at 4° C. until the culture turned an intensered colour and dye production was high (usually 3 to 5 days).

[0074] Once the culture produced sufficient dye for harvesting, theculture medium, including the fungal biomass was transferred intoethanol at a ratio of one volume of culture medium to two volumes ofethanol. The dye was extracted into the ethanolic phase by incubation at4° C. and shaking for 16 hours. The liquid phase was decanted from theresidual culture medium, and centrifuged at 3000 rpm for 10 min at 4° C.

[0075] The clarified extract (Extract A) was either stored for use, orpurified further according to one of the procedures described underexamples 2 and 3.

Example 2

[0076] Purification of Extract A to Produce Extract B.

[0077] The crude ethanolic extract of Extract A produced according tothe method described in example 1 was reduced in volume under highvacuum, chromatographed on cellulose powder using methanol as a solventand applied to a sephadex LH-20 column, also eluted with methanol.Fractions were collected over 48 hours and the purple band eluting nearthe end was collected. This fraction was freeze dried and resuspended ina minimum volume of (0.1% acetic acid) methanol and stored.

Example 3

[0078] Purification of 5,6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione.

[0079] Extract B produced according to the method described in example 2was subjected to HPLC purification (Supelco C16-amide column, resolvedin 75% methanol/water, 0.05% acetic acid, then 50% acetonitrile/water,0.05% acetic acid). The final fraction was frozen (−20° C.) to removewater and the remaining acetonitrile was evaporated under a stream ofnitrogen.

[0080] This procedure produced analytically pure, 6-dihydro-3-[(1Z, 4E,6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dionewhich was an orange solid.

Example 4

[0081] Structure Determination of ,6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione.

[0082] The compound produced according to example 3 was analysed using acombination of NNR spectroscopy and high resolution mass spectrometry todetermine the structure of, 6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione.

[0083] Nuclear Magnetic Resonance Spectroscopy.

[0084] The NMR sample was prepared by dissolving the HPLC purifiedcompound obtained from Example 3 (˜5 mg) in CDCl₃ (0.5 mL; 99.96 atom %,Aldrich) and filtering it into an NMR tube (PP527, Wilmald). The samplewas degassed and equilibrated under an atmosphere of nitrogen.

[0085] The NMR data were acquired on a Bruker DRX600 (600 MHz) NMRspectrometer at 27° C. and processed using xwinNMR (version 2.6;Bruker). All 2D NMR experiments were run with quadrature detection withan ¹H spectral width of 6009 Hz and a recycle delay of 2 s. Chemicalshifts were referenced to the residual CHCl₃ (dH 7.26 ppm; dC 77.01ppm). High power ¹H p/2 pulses were determined to be 9.5 ms and lowpower (for MLEV spin lock) at 25.15 ms. ¹³C high power p/2 pulse was10.5 ms and a low power pulse of 65 ms was used for GARP decoupling.Gradient pulses were delivered along the z-axis using a 100 step sineprogram.

[0086] Data for 1D experiments were acquired using 32 K real points andzero filled to 64 K and then Gaussian multiplied for resolutionenhancement. Carbon-hydrogen correlation (HSQC) was achieved via asensitivity enhanced double INEPT transfer using echo/antiecho-TPPIgradient (80:20.1) selection (Palmer, A. G., III, Cavanagh, J., andWright, P. (1991) J. Magn. Reson. 93, 151-70; Schleucher, J.,Schwendinger, M., Sattler, M., and Schmidt, P. (1994) J. Biomol. NMR 4,301-6; Kay, L. E., Keifer, P., and Saarinen, T. (1992) J. Am. Chem. Soc.114(26), 10663-5). 2K data points were collected in t2 (128 scans perincrement) with a 1.3 s recycle delay with decoupling duringacquisition. In t1, 512 increments were used (10-170 ppm) and the INEPTsequence was optimized for a X-H coupling of 145 Hz. A gradient ratio of80:20.1 was used to select echo/antiecho-TPPI phase sensitivity.

[0087] One dimensional ROESY spectra were measured using a selectiveGaussian pulse on the proton of interest (Kessler, H., H. Oschkinat, C.Griesinger & W. Bermel, (1986) J. Magn. Reson. 70, 106; Stonehouse, J.,P. Adell, J. Keeler & A. J. Shaka, (1994) J. Am. Chem. Soc. 116, 6037;Stott, K., J. Stonehouse, J. Keeler, T. L. Hwang & A. J. Shaka, (1995)J. Am. Chem. Soc. 117, 4199 4200). A 1000 step Gaussian program (60 ms,64.6 dB) was used to achieve a p/2 pulse. A mixing time of 100 ms (13dB) was used for a continues wave spin lock. Gradient selection wasachieved with a 15% gradient along the z-axis. 10K transients wereaccumulated over 6009 Hz. ROE enhancements were measured as a percentageof the irradiated peak and not compensated for offset from the carrierfrequency.

[0088] Two dimensional homonuclear Hartman-Hahn transfer spectra (TOCSY)were measured using the MLEV17 (Bax, A., & Davis, D. G. (1985) J. Magn .Reson. 63(1), 207-13) pulse sequence flanked with 2 ms low power trimpulses. Sine bell shifted (90°) apodisation was used in the processingof both dimensions.

[0089] 2D ¹H-¹³C correlation via heteronuclear zero and double quantumcoherence optimized for long range couplings (HMBC) with low-passJ-filter (145 Hz) to suppress one-bond correlations (Bax, A., & M. F.Summers, (1986) J. Am. Chem. Soc. 108, 2093-2094) was acquired with nodecoupling during acquisition time and using gradient pulses(50:30:40.1) for selection. The delay for evolution of long rangecouplings was optimized for couplings of 20, 10, 5 and 2 Hz in separateexperiments. A spectral width of 210 ppm was used in F1 and the finalspectrum magnitude calculated to destroy phase information.

[0090]¹H NMR (600 MHz, CDCl₃) δ1.75, s, C9a-Me; 1.84, dd, J 6.7, 1.1 Hz,C9′-Me;2.80, dd, J 17.2, 3.6 Hz, H5α; 2.89, ddd, J 17.1, 11.5, 1.9 Hz,H5β; 3.85, dd, J 12.2, 5.5 Hz, C6CH ₂OH; 3.97, dd, J 12.2, 3.4 Hz, C6CH₂OH; 4.39, m, H6; 5.97, m, H9′; 6.10, d, J 15.1 Hz, H4′, 6.19, ddd, J15.1, 10.6, 1.6 Hz, H8′; 6.25, dd, J 14.6, 11.2 Hz, H6′; 6.58, dd, J14.5, 10.5 Hz, H7′; 6.79, s, H2′; 7.16, bs, H4, 7.32, dd, J 15.2, 11.2Hz, H5′; 7.85, s, H8.

[0091]¹³C NMR (125 MHz, CDCl₃) δ 18.8, C9′-Me; 28.0, C9a-Me; 29.3, C5;63.4, C6-CH₂OH; 79.0, C6; 86.2, C9a; 101.0, C2′; 112.2, C8a; 113.4, C4;115.3, C3; 126.5, C4′; 128.6, C6′; 131.7, C8′; 136.0, C9′; 140.9, C4a;142.0, C7′; 142.6; C5′; 159.0, C8; 168.2, C2/C3a; 177.7, C1′; 185.0,C3′; 190.0, C9.

[0092] Heteronuclear single quantum coherence (HSQC) was used tocorrelate and assign all protons to protonated carbons. In addition,each spin system was characterised by running a series of totalcorrelation spectroscopy (TOCSY) experiments with mixing times of 8msec, 20 msec, 100 msec. The shortest mixing time gave correlations toonly the directly coupled protons whereas the longest mixing timeidentified the entire spin system and gave information about very smalllong range couplings which were valuable for assigning the positions ofthe many (apparently) uncoupled protons. Thorough space connectivitieswere achieved by a series of one dimensional selective rotating framecorrelation spectroscopy (ROESY) experiments. Selective excitation wasachieved by a low power Gaussian 90 degree pulse and using gradientsection to observe only ROE's free of TOCSY transfers. A mixing time of100 msec was found to be optimal. Long range heteronuclear multiplequantum coherence (HMBC) spectra with mixing times of 25, 50, 100 and250 msec was found to be essential to assign all non-protonated carbons.Even after this, C2 was found not to correlate with any protons and wascoincident with C3a at 168.2 ppm.

[0093] From the spectral data, several tricyclic skeletons weretheoretically possible but one (6-dihydro-3-[(1Z, 4E, 6E,8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione),fitted the available data exactly.

[0094] Mass Spectrometry

[0095] The compound produced according to Example 3 was subjected tohigh resolution mass spectrometry to determine that the molecularformula of the compound was C₂₃H₂₂O₇.

[0096] Mass spectrum (ESI, positive ion) m/e 411 (M+H⁺, 55%), 317 (5),249 (12), 163 (10), 121 (100), 93 (4). Negative ion ESI-FTICR Found:M-H⁺, 409.1304, C₂₃H₂₁O₇ requires 409.1293; found: 247.0617, C₁₃H₁₂O₅requires 247.0685.

[0097] λ_(max) and ν_(max)

[0098] λ_(max) (MeOH) 432, 555 nm, ε 10000, 4000. λ_(max)(alkaline MeOH)432 nm, ε 16000. λ_(max) (acidic MeOH) 390, 560 nm, ε6000, 10000.

[0099] ν_(max) (neat) 3400 (br), 2925, 2854, 1744, 1712, 1589, 1259,1010 cm⁻.

Example 5

[0100] Protein Gel Staining Using Extract A.

[0101] A protein gel was prepared according to any one of a number ofstandard protocols. For example, an SDS page gel was prepared accordingto the protocol of Laemrnli (U.K. 1970, Nature, 227:680-685). Afterelectrophoresis was completed, the gel was removed from theelectrophoresis apparatus. The gel was fixed in a container containing100 mL of a staining solution composed of 90 mL water, 10 mL glyceroland 5 mL of Extract A. The gel and staining solution were incubated withgentle shaking for 90 minutes. After the 90 minute staining procedure,the solution was removed and the gel briefly washed three times inwater. After these brief washes, the gel was placed in 100 mL of 10%glycerol in water and incubated with shaking for a further 30 min.

[0102] To visualise the proteins in the gel, the gel was transferred toa UV transilluminator (307 nm), and photographed using polaroid 667black and white film and a filter set designed for the detection ofethidium bromide gels (eg. Molecular probes E-7591).

Example 6

[0103] Epifluoroescence Differentiation of Sporulated and VegetativeCells.

[0104] A micro-organism is cultured under conditions to promotesporulation and Extract B is used to facilitate the identification ofsporulated cells within a predominantly vegetative cell culture. Forexample, Saccharomyces cerevisiae was grown in a water based brothcontaining 10 g/L Glucose, 5 g/L yeast extract, and 10 g/L peptone.After a 16 hour incubation at 30° C., the cells were harvested bycentrifugation and resuspended in 1 mL of water. The 5 μL aliquots ofthe culture were spotted onto a semi-solid medium composed of 5%Potassium acetate, 2% Agar and water. The cultures were incubated at 22°C. for 4 days to allow sporulation of the yeast cells. The sporulatedculture was resuspended in water to a density of 1×10⁹ cells per mL. 5μL of Extract B was added to 1 mL of the sporulated culture. Tocounterstain, 5 μL of a 10 mM solution (in DMSO) of5(6)-carboxyfluorecein diacetate (CFDA), was added to the sporulatedculture. After 5 minutes, the sporulated culture was harvested bycentrifugation and resuspended in 1 mL of water. Examination under anepifluorescence microscope (excitation 488 nm) allowed for rapiddifferentiation between sporulated and vegetative cells.

[0105] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

1. A compound according to formula (I):

wherein: each of R, R′ and R″ is a hydrogen atom, halogen atom or astraight or branched chain C₁₋₂₀ alkyl, alkenyl or alkynyl groupoptionally substituted with one or more halogen, hydroxy and/or oxygroup; rings A, B and C optionally include one or more double bonds;rings B and C are optionally substituted with one or more halogen atoms,including tautomers of formula (I), and the compound is capable ofinteracting with an organic compound and when interacting with theorganic compound, the compound emits fluorescence after excitation at abroad range of wavelengths.
 2. A compound as claimed in claim 1according to the formula (Ia), including tautomers thereof:


3. A compound as claimed in claim 2 having the stereochemistry asdepicted in formula (Ib), including tautomers thereof:


4. A compound as claimed in any one of claims 1 to 3 wherein R is astraight or branched chain C₁₋₂₀ conjugated alkenyl group optionallysubstituted with hydroxy and oxy groups; R′ is a straight or branchedchain C₁₋₂₀ alkyl group optionally substituted with a hydroxy group andR″ is a straight or branched chain C₁₋₂₀ alkyl group, includingtautomers thereof.
 5. A compound as claimed in 3 wherein R is—C(OH)CHC(O)(CH)₆CH₃, R′ is —CH₂OH and R′ is ′Me being the compound5,6-dihydro-3-[(1Z, 4E, 6E, 8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dioneaccording to formula (Ic), including tautomers thereof:


6. A compound according to formula (I):

wherein each of R, R′ and R″ is a hydrogen atom, halogen atom or astraight or branched chain C₁₋₂₀ alkyl, alkenyl or alkynyl groupoptionally substituted with one or more halogen, hydroxy and/or oxygroups; ring A, B and C are optionally substituted with one or morehalogen atoms; and rings A, B and C optionally include one or moredouble bonds; with the proviso that: (i) R≠—C(OH)CHC(O)(CH)₆C(Et)(Me)when R′=—CH₂OH, R″=Me, ring A includes a double bond between C3 and C3a;ring B includes a double bond between C4 and C4a; and ring C includes adouble bond between C8 and C8a; (ii) R≠—C(OH)CHC(O)(CH)₆C(Et)(Me) whenR′=Me, R″=Me, ring A includes a double bond between C3 and C3a; ring Bincludes a double bond between C4 and C4a; and ring C includes a doublebond between C8 and C8a; (iii) R≠—C(OH)CHC(O)(CH)₆C(Et)(Me) when R′=Me,R″=Me, ring A includes a double bond between C3 and C3a; ring B includesa double bond between C4 and C4a; and ring C includes a double bondbetween C8 and C8a and C5 and C6; (iv) R≠—C(O)(CH)₄Me when R′=-n-propyl,R″=Me, ring A includes a double bond between C3 and C3a; ring B includesa double bond between C4 and C4a; and ring C includes a double bondbetween C8 and C8a and C5 and C6; (v) R≠—C(O)(CH)₆Me when R′=-n-propyl,R″=Me, ring A includes a double bond between C3 and C3a; ring B includesa double bond between C4 and C4a; and ring C includes a double bondbetween C8 and C8a and C5 and C6; (vi) R≠—C(O)(CH)₅Me when R′=—(CH)₂Me,R″=Me, ring A includes a double bond between C3 and C3a; ring B includesa double bond between C4 and C4a; and ring C includes a double bondbetween C8 and C8a and C5 and C6; (vii) R≠—C(O)(CH)₆Me when R′=—(CH)₂Me,R″=Me, ring A includes a double bond between C3 and C3a; ring B includesa double bond between C4 and C4a; and ring C includes a double bondbetween C8 and C8a and C5 and C6; (viii) R≠—(CH)₂C(Me)CHC(Me)(Et) whenR′=—Ac, R″=Me, C4=Cl, ring A includes a double bond between C3 and C3a;ring B includes a double bond between C4 and C4a; and ring C includes adouble bond between C8 and C8a and C5 and C6; (ix)R≠—(CH)₂C(Me)CHC(Me)(Et) when R′=—Ac, R″=Me, ring A includes a doublebond between C3 and C3a; ring B includes a double bond between C4 andC4a; and ring C includes a double bond between C8 and C8a and C5 and C6;(x) R≠—(CH)₂C(Me)CH-i-Pr when R′=—Ac, R″=Me, ring A includes a doublebond between C3 and C3a; ring B includes a double bond between C4 andC4a; and ring C includes a double bond between C8 and C8a and C5 and C6;(xi) R≠—C(O)(CH)₄Me when R′=—(CH)₂Me, R″=Me, ring A includes a doublebond between C3 and C3a; ring B includes a double bond between C4 andC4a; and ring C includes a double bond between C8 and C8a and C5 and C6;(xii) R≠—C(O)(CH)₄Me when R′=-n-Pr, R″=Me, ring A does not include adouble bond; and rings B and C include double bonds between C8a and C4aand C5 and C6; (xiii) R≠—C(O)(CH)₄Me when R′=-n-Pr, R″=Me, ring A, B andC do not include double bonds; (xiv) R≠—C(O)(CH)₆Me when R′=—(CH)₂Me,R″=Me, ring A does not include a double bond; and rings B and C includedouble bonds between C8a and C4a and C5 and C6; (xv) R≠—C(O)(CH)₄Me whenR′=—C(CH₂)(Me), R″=Me, ring A does not include a double bond; and ringsB and C include double bonds between C8a and C4a and C5 and C6; (xvi)R≠—C(O)(CH)₄Me when R′=—(CH)₂Me, R″=Me, ring A does not include a doublebond; and rings B and C include double bonds between C8a and C4a and C5and C6, including tautomers thereof.
 7. A compound as claimed in claim 6according to the formula (Ia):


8. A compound as claimed in claim 7 having the stereochemistry asdepicted in formula (Ib):


9. A compound as claimed in any one of claims 1 to 8, wherein thecompound when interacting with an organic compound, emits fluorescenceafter excitation at blue wavelengths.
 10. A compound as claimed in claim9 wherein the compound emits fluorescence when excited by light in therange 300-560 nm.
 11. A compound as claimed in claim 10 wherein theorganic compound is a protein and the compound when interacting with theprotein emits fluorescence after excitation at 307 nm.
 12. A compound asclaimed in claim 10 wherein the compound emits fluorescence when excitedby light at about 400 nm, 488 nm and 514 nm.
 13. A compound as claimedin claim 12 wherein compound emits fluorescence at 600 nm afterexcitation at 488 nm.
 14. A composition including a compound as claimedin any one of claims 1 to 13, an analytically acceptable carrier andoptionally, a flurochrome.
 15. A process for the preparation of acompound as claimed in any one of claims 1 to 13 comprising culturing afungal species under conditions such that the fungal species producesthe compound, and separating the compound from the culture.
 16. Use of acompound as claimed in any one of claims 1 to 13 as a fluorescent dye ormarker.
 17. Use as in claim 16 in a technique for staining, labellingand/or detecting organic molecules.
 18. A method for fluorescentlabelling an organic compound, comprising causing a compound as claimedin any one of claims 1 to 13 to bind to an organic compound in a mannersuch that the organic compound is fluorescently labelled with thecompound.
 19. A compound, method, composition or use as claimed in anyone of claims 1 to 14 and 16 to 18, wherein the organic compound is aprotein, peptide, sugar, nucleic acid, cell surface molecule ordetergent, or is included in an antibody or a cell.
 20. A compoundmethod or use as in claim 19 wherein the compound is bound to theorganic compound through a linking molecule.
 21. A method of detecting aorganic compound in a sample including the organic compound, the methodcomprising labelling the organic compound according to the method ofclaim 18, and detecting the organic compound in the sample by monitoringor detecting its fluorescence.
 22. A method as claimed in claim 21wherein the labelled organic compound is detected when the sample isexposed to light from a wide range of wavelengths, preferablywavelengths in the range of 300-560 nm, more preferably bluewavelengths.
 23. A method as claimed in claim 21 or claim 22 wherein themonitoring or detecting of the fluorescence of the labelled-organiccompound is by transillumination, spectroscopy, microscopy or cytometry.